Method for generating primate trophoblasts

ABSTRACT

The first method to cause a culture of human and other primate stem cells to directly and uniformly differentiate into a committed cell lineage is disclosed. Treatment of primate stem cells with a single protein trophoblast induction factor causes the cells to transform into human trophoblast cells, the precursor cells of the placenta. Several protein factors including bone morphogenic protein 4 (BMP4), BMP2, BMP7, and growth and differentiation factor 5 can serve as trophoblast-inducting factors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/365,136 filed Mar. 15, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

Modern cell biology includes a variety of techniques to manipulatevarious cells of living organisms in vitro. Of particular interest is acategory of cell known as a stem cell. Stem cells are undifferentiatedor only partially differentiated cells that have the capability todifferentiate into a number of progenitor and mature cell lineages andtypes. The term “stem cells” can be used to refer to a cell type whichis the progenitor of a differentiation cellular lineage in a largerorganism, such as hematopoietic stem cell, or can refer to totallyundifferentiated stem cells which, at least in theory, have the abilityto differentiate into any of the tissues of the body. Stem cells are, ata minimum, pluripotent, meaning that they have the potential todifferentiate into many different cell types, and may be totipotent,meaning have the potential to differentiate into any cell type of amature organism of the species. Stem cell cultures have been developedfrom a variety of tissue types and from a number of different animals.

Recently, it has become possible to generate, culture and maintaincultures of primate embryonic stem cells, including human and rhesusembryonic stem cells. See, for example, U.S. Pat. No. 5,843,780 and No.6,200,806 to Thomson. Primate embryonic stem cells are stem culturescreated from embryos that survive indefinitely in culture anddemonstrate the ability to differentiate into the major tissue types ofthe primate body. Primate embryonic stem cells can be maintainedindefinitely in an undifferentiated state in culture, or can be allowedto start a differentiation process by which various of the cells becomecommitted to one or multiple developmental lineages. Typically, thedifferentiation of stem cells into different tissue types begins withthe creation of embryoid bodies, which causes the stem cells in theembryonic body to begin to differentiate into various cell types.

A more differentiated type of human cell of scientific and researchinterest is a human trophoblast. A trophoblast is a cell which is aprecursor of the cells which participate in the formation of the humanplacenta. When an embryo begins differentiation, at the stage of ablastocyst, the cells in the inner cell mass are committed to form thecells which will become the embryo, while the outer cells of theblastocyst become committed to participate in the development of theplacenta. Trophoblast cells have been isolated before, but they aredifficult to isolate and have not been available for research insignificant amounts. Mouse trophoblast cell lines have been created fromblastocyst and post-implantation trophoblasts. Human trophoblast celllines have been created from transformed placental cells, but techniquesto create cultures of primate trophoblasts from embryonic cells or stemcell lines have not yet been reported. While human embryonic stem cellswill spontaneously differentiate into a number of differentiated celltypes, including some trophoblast cells, this phenomenon has not led tothe creation of useful cultures of trophoblast cells. In fact, mouseembryonic stem cells appear to lack the ability to differentiate intotrophoblast, and hence, the supply of trophoblasts has always beenextremely limited. A replenishable supply of consistent trophoblastcells would be very useful for many pharmaceutical investigations. Inparticular, the exploration of contraceptive drugs targeting embryoimplantation and therapeutics preventing placenta-related birth defectsremain the topics of scientific investigation that can be pursued withmore ease provided that a source of primate trophoblasts is available.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that a method to induce primatestem cells to predominantly differentiate into human trophoblastsincludes the step of culturing the primate stem cells in the presence ofa protein trophoblast-inducing factor.

The present invention is also directed to uniform cultures of primatetrophoblast cells created by the method taught here.

The present invention is also directed to a method for testing agents onplacental cells in which the agents are exposed to trophoblast culturesas described here.

It is an object of the present invention to enable the creation ofcultures of nearly pure trophoblasts in a uniform consistent andreproducible manner.

It is a feature of the present invention in that it teaches the firstmethod known to cause primate stem cells in culture to repeatedly,directly, individually and in synchrony predominantly differentiate intoa committed cell lineage.

Other objects, features and advantages of the present invention willbecome apparent from the following specification.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a graphical illustration of the secretion of hormones bytrophoblast cells cultured according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is premised on an observation. It has been foundby the inventors here that certain protein factors will cause primateembryonic stem cells to differentiate directly into trophoblast cells.The trophoblast cells are stable and exhibit all of the cellularcharacteristics of placental precursor cells. Since the creation andculture of primate and human embryonic stem cells have becomestandardized and readily reproducible, this observation makes possiblefor the first time the creation of a major class of a single cell type(trophoblast cells) directly from a human or other primate stem cellsource, without intervening creation of an embryoid body. It has beenfound here that the protein factors that cause direct differentiation ofprimate embryonic stem cells to trophoblast cells include bonemorphogenic protein 4 (BMP4) as well as related protein factors such asBMP2, BMP7, and growth and differentiation factor 5 (GDF5). Such afactor is here referred to as a trophoblast-inducing factor.

The availability of human and other primate trophoblast cells inreproducible quantities makes possible many investigative studies on thebehavior of placental cells. It is now possible to have a reproducibleand inexhaustible supply of placental precursor cells. In particular, itis envisioned that the trophoblast cell cultures can be used forchemical reaction studies to model the behavior of placental cells foruse in drug testing, both for general toxicology as well as for specificeffects on placental cells. For example, agents, which would inhibitfertilized embryo implantation in a uterus, i.e. birth control agents,can be investigated by observing the effects of putative agents on thetrophoblast cell cultures.

This disclosure includes data which demonstrates that human and otherprimate stem cells can be directly transformed into trophoblast cells bytreatment with a single protein factor. As used herein, primate stemcells refers to human or other primate undifferentiated cells which areat least pluripotent. The stem cells used in the examples beloworiginate from human and rhesus embryos, and hence are known as primateembryonic stem cells. Embryonic stem cells are stem cells derived fromhumans at some stage of development. The method described here is,however, equally applicable to human stem cells derived from otherorigins, including embryonic germ line cells and stem cells isolatedfrom mature primate bodies. Note that the fact that human stem cellswill differentiate into trophoblast cells is unexpected based onexperience with mouse stem cells. Efforts to derive trophectoderm tissuefrom mouse stem cells by manipulation of the external cultureenvironment have so far been unsuccessful, and when formed into chimeraswith intact pre-implantation embryos, mouse stem cells rarely contributeto the trophoblast. The failure of mouse embryonic stem cells to formtrophoblast cells is consistent with the theory that mouse embryonicstem cells are developmentally similar to primitive ectoderm, whichforms after delamination of the primitive endoderm from the inner callmass and which no longer contributes to the trophoblast lineage. Theability of human embryonic stem cells to form trophoblast cells suggestsa basic difference between the development potential of mouse and humanembryonic stem cells.

Note that the method here involves the application of thetrophoblast-inducing factor directly to stem cells in culture, withoutany intervening processes normally associated with differentiation ofstem cells. In particular, note that no stage of transition to embryoidbodies is associated with this method. The differentiation process forstem cells cultured by other means is generally not uniform, in thesense that many different cell lineages or cell types normally result.By contrast, the method described here results in a mass differentiationof the stem cells to a common differentiated cell type, trophoblast.

To demonstrate that the directed differentiation of stem cells intotrophoblast cells is due to the influence of the trophoblast-inducingfactor, it is possible to inhibit the action of the trophoblast-inducingfactor and observed the result. If BMP4 is the trophoblast-inducingfactor, this protein can be inhibited by soluble BMP4 receptor or by theantagonizing protein noggin. That is, if one cultures primate stem cellswith BMP4 only, the stem cell culture will exhibit large scale directeddifferentiation to trophoblast cell types. However, if one cultures asimilar primate stem cell culture with BMP4 and an inhibitor, such asthe soluble BMP4 receptor or noggin, the differentiation to trophoblastcells will not occur.

Human embryonic stem cells in culture have a very distinctivemorphology. The cells are small, compact, and uniform, have distinctcell membranes and cluster in groups. The differentiation of stem cellsinto other cell types is a visible process as the stem cells becomelarger and more diffuse. Experienced technicians can recognize bycellular appearance of the differentiated cells for many cell types. Inthe case of trophoblast cells, the cells do become larger and flatten,and the cells membranes becomes diffuse to invisible. However, tosupplement the status of the trophoblast cells, various characterizingstudies of the cells were undertaken. A gene expression study using DNAmicroarrays was conducted to examine the gene expression pattern of thecells. The secretion of placental hormones by the cells was alsoexamined. The results were consistent with the identification of thesedifferentiated cells as trophoblast cells. This confirmed that themorphological identification of these cells was correct.

EXAMPLES

A human embryonic stem cells line, H1, was cultured on aMatrigel™-coated plastic plate in medium that had been conditioned onmouse embryonic fibroblasts (MEF) and supplemented with basic fibroblastgrowth factor (bFGF) at 4 mg/ml. Human bone morphogenic protein 4 (BMP4)(R&D Systems, Minneapolis, Minn., also the source for other recombinantproteins listed here) was applied to the stem cells at concentrations of1, 10 and 100 ng/ml of culture medium. The stem cells were as amonolayer and not aggregated in embryoid bodies. The H1 cells thenunderwent a dose and time dependent morphological change, becomingspread out, flat, thin and enlarged or elongated with their nucleibecoming smaller. These changes are consistent with the morphology oftrophoblast cells. The morphological changes began with the cells at theedge of each colony and spread inward from there. The changingmorphology became evident on day 2 for cultures treated with 100 ng/mlBMP4, day 3 or 4 for cultures treated with 10 ng/ml and days 4 to 5 forcultures treated with 1 ng/ml BMP4.

Similar experiments were conducted with other members of the BMP4protein signal family. Proteins which have been demonstrate to activatea similar effect, to cause stem cells to change to trophoblast cells,include BMP2, BMP7, and growth and differentiation factor 5(GDF5). Otherproteins, including members of the TGF superfamily, such as TGF beta 1and activin, were found not to activate this same morphological changein stem cells. Similar morphological changes were observed on rhesusembryonic stem cell lines treated with BMP4, BMP2, BMP7 and GDF5.

The change in morphology of the stem cells treated with thetrophoblast-inducing factor is consistent with the morphological changesthat occur in the development of an embryo where some cells becomecommitted to a lineage resulting in the placenta. In addition to themorphological changes, the cells begin to express transcription factorsGATA2 and GATA3 and chorionic gonadatrophin alpha and beta genes, all ofwhich are expressed in trophoblasts created by other means. The cellsproduce high amounts of placental hormones including chorionicgonadatrophins, estradiol, and progesterone. The cells continue tosecrete these hormones indefinitely. Flow cytometry of the cellsdemonstrate that the cells are, at least predominantly, CG betapositive.

Experiments were also conducted in which antagonists of BMP familyfactors were added to the culture at the same time as the proteinfactor. It was found that if a soluble BMP receptor (at 100 ng/ml) orthe BMP antagonizing protein noggin (at 300 ng/ml) were added to theculture at the same time as for BMP4, the morphological change in thestem cells was entirely prevented. This demonstrates the specificity ofthe effect of the activating protein factor.

Similar experiments were conducted on another stem cell lines, named H9,with similar result in the production of trophoblast cells. In addition,a similar experiment was conducted on H1 cells cultured in the absenceof bFGF, suggesting that the effect is universal to human stem cellsfrom various donors and is not dependent on the presence of bFGF.

To further investigate the character of the trophoblasts, cDNAmicroarrays were used to analyze genes differentially expressed in theBMP4-treated cells and the untreated undifferentiated H1 cells. Of the43,000 cDNA genes examined on the arrays, a cluster of only 19 clones,representing 14 genes, was strongly upregulated during all the timepoints examined. Of these 14 genes, 11 have been previouslycharacterized as related to the development of trophoblast or placenta.Many of these genes encode transcription factors, such as transcriptionfactor AP-2 (TFAP2), msh homeobox homolog 2 (MSX2), a suppressor ofcytokine signaling 3 (SSI3), GATA binding proteins 2 and 3 (GATA2 andGATA3) and hairy/enhancer-of-spli related with YPRW motif 1 (HEY1). Byday 7 of treatment with BMP4, there was also observed a dramaticincrease in mRNA expression of many genes known to be expressed introphoblast or placenta, such as genes encoding CG-α and CG-β subunits,luteinizing homone-alpha and placental growth factor. We also suedRT-PCR to observe enhanced expression of trophoblast markers, includingCG-β, glial cells missing-1 (GCM1), the non-classical HLA class Imolecule HLA-G1, and CD9. All of the top ten upregulated clones,representing 8 genes, in the microarray analysis, with one exception,encode proteins or peptides previously associated with genes expressedin trophoblast cells. By contrast, after 7 days of BMP4 treatment,transcripts of several genes highly expressed in pluripotent cells haddeclined, such as those encoding the POU domain, class 5, transcriptionfactor 1 (POU5F1, also known as OCT4), and telomerase reversetranscription factor (TERT).

To further confirm the character of the cells, the amount of theplacental hormones CG, estradiol and progesterone secreted into themedium of the cells was examined. H1 cells treated with BMP4 showedmarkedly higher concentrations of each hormone as compared toundifferentiated cells or cells differentiated in unconditioned medium.FIG. 1 illustrates the time course in the increase of these hormones inthe cells exposed to BMP4 (CM+BMP4) as compared to cells without BMP4(CM) and cells permitted to differentiate in unconditioned medium (UM).

1-5. (canceled)
 6. An in vitro culture comprising a replenishablepopulation of primate trophoblast cells derived from undifferentiatedprimate stem cells exposed to a trophoblast inducing factor, wherein thefactor is selected from the group consisting of bone morphogenic protein4 (BMP4), bone morphogenic protein 2(BMP2), bone morphogenic protein7(BMP7) and growth and differentiation factor 5(GDF5), and wherein thestem-cell derived trophoblast cells express chorionic gonadotropin andare derived directly from undifferentiated cells without passing throughan embryoid body stage.
 7. (canceled)
 8. The culture of claim 6 whereinthe trophoblast cells are human cells.
 9. The culture of claim 6 whereinthe trophoblast cells are non-human primate cells.
 10. A method oftesting the effect of an agent on cells of the primate trophoblastlineage comprising: (a) culturing undifferentiated primate stem cells ina culture medium comprising a protein trophoblast inducing factorselected from the group consisting of bone morphogenic protein 4 (BMP4),bone morphogenic protein 2(BMP2), bone morphogenic protein 7(BMP7) andgrowth and differentiation factor 5 (GDF5); (b) exposing the cells ofstep (a) to the agent; and (c) observing the effects of the agent on thetrophoblast cells.
 11. (canceled)
 12. The method of claim 10 wherein thetrophoblast-inducing factor is applied to the stem cells at aconcentration of between 1 and 100 nanograms per milliliter of culturemedium.
 13. The method of claim 10 wherein the undifferentiated stemcells are human embryonic stem cells.
 14. (canceled)
 15. the method ofclaim 10 wherein the agent is a contraceptive or a birth control agent.16. the method of claim 10 wherein the agent is a therapeutic forpreventing placenta-related birth defects.
 17. An in vitro culturecomprising a replenishable population of primate trophoblast cellsderived from undifferentiated primate stem cells exposed to atrophoblast inducing factor, wherein the factor is selected from thegroup consisting of bone morphogenic protein 4 (BMP4), bone morphogenicprotein 2(BMP2), bone morphogenic protein 7(BMP7) and growth anddifferentiation factor 5(GDF5), and wherein the stem-cell derivedtrophoblast cells are mononuclear, express chorionic gonadotropin andare derived directly from undifferentiated cells without passing throughan embryoid body stage.